Differential substrate specificity and kinetic behavior of Escherichia coli YfdW and Oxalobacter formigenes formyl coenzyme A transferase.

The yfdXWUVE operon appears to encode proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions. Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, findings from structural genomic studies have shown that the structure of YfdW, the protein encoded by the yfdW gene, is homologous to that of the enzyme that mediates oxalate catabolism in the obligate anaerobe Oxalobacter formigenes, O. formigenes formyl coenzyme A transferase (FRC). We now report the first detailed examination of the steady-state kinetic behavior and substrate specificity of recombinant, wild-type YfdW. Our studies confirm that YfdW is a formyl coenzyme A (formyl-CoA) transferase, and YfdW appears to be more stringent than the corresponding enzyme (FRC) in Oxalobacter in employing formyl-CoA and oxalate as substrates. We also report the effects of replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the E. coli protein. The results of these experiments show that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Our findings illustrate the utility of structural homology in assigning enzyme function and raise the question of whether oxalate catabolism takes place in E. coli upon the up-regulation of the yfdXWUVE operon under acidic conditions.

Reinvestigation of the catalytic mechanism of formyl-CoA transferase, a class III CoA-transferase.

Formyl-coenzyme A transferase from Oxalobacter formigenes belongs to the Class III coenzyme A transferase family and catalyzes the reversible transfer of a CoA carrier between formyl-CoA and oxalate, forming oxalyl-CoA and formate. Formyl-CoA transferase has a unique three-dimensional fold composed of two interlaced subunits locked together like rings of a chain. We here present an intermediate in the reaction, formyl-CoA transferase containing the covalent beta-aspartyl-CoA thioester, adopting different conformations in the two active sites of the dimer, which was identified through crystallographic freeze-trapping experiments with formyl-CoA and oxalyl-CoA in the absence of acceptor carboxylic acid. The formation of the enzyme-CoA thioester was also confirmed by mass spectrometric data. Further structural data include a trapped aspartyl-formyl anhydride protected by a glycine loop closing down over the active site. In a crystal structure of the beta-aspartyl-CoA thioester of an inactive mutant variant, oxalate was found bound to the open conformation of the glycine loop. Together with hydroxylamine trapping experiments and kinetic as well as mutagenesis data, the structures of these formyl-CoA transferase complexes provide new information on the Class III CoA-transferase family and prompt redefinition of the catalytic steps and the modified reaction mechanism of formyl-CoA transferase proposed here.

Determination of oxalyl-coenzyme A decarboxylase activity in Oxalobacter formigenes and Lactobacillus acidophilus by capillary electrophoresis.

Oxalyl-coenzyme A decarboxylase (OXC) is a key enzyme in the catabolism of the highly toxic oxalate, catalysing the decarboxylation of oxalyl-coenzyme A (Ox-CoA) to formyl-coenzyme A (For-CoA). In the present study, a capillary electrophoretic (CE) method was proposed for the assessment of the activity of recombinant OXC from two bacteria, namely Oxalobacter formigenes DSM 4420 and Lactobacillus acidophilus LA 14. In particular, the degradation of the substrate Ox-CoA occurring in the enzymatic reaction could be monitored by the off-line CE method. A capillary permanently coated with polyethylenimine (PEI) was used and in the presence of a neutral background electrolyte (50 mM phosphate buffer at pH 7.0), a reversal of the electroosmotic flow was obtained. Under these conditions, the anodic migration of Ox-CoA (substrate) and For-CoA (reaction product) occurred and their separation was accomplished in less than 12 min. The CE method was validated for selectivity, linearity (range of Ox-CoA within 0.005-0.650 mM), sensitivity (LOD of 1.5 microM at the detection wavelength of 254 nm), precision and accuracy. Steady state kinetic constants (V(max), K(m) or k') of OXC were finally estimated for both the bacteria showing that although L. acidophilus LA 14 provided a lower oxalate breakdown than O. formigenes DSM 4420, it could be a potentially useful probiotic in the prevention of diseases related to oxalate.

Detection and characterization of merohedral twinning in crystals of oxalyl-coenzyme A decarboxylase from Oxalobacter formigenes.

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate dependent enzyme active in the catabolism of the highly toxic compound oxalate. The enzyme from Oxalobacter formigenes has been expressed as a recombinant protein in Escherichia coli, purified to homogeneity and crystallized. Two crystal forms were obtained, one showing poor diffraction and the other merohedral twinning. Crystals in the former category belong to the tetragonal space group P4(2)2(1)2. Data to 4.1 A resolution were collected from these crystals and an incomplete low resolution structure was initially determined by molecular replacement. Crystals in the latter category were obtained by co-crystallizing the protein with coenzyme A, thiamin diphosphate and Mg(2+)-ions. Data to 1.73 A were collected from one of these crystals with apparent point group 622. The crystal was found to be heavily twinned, and a twin ratio of 0.43 was estimated consistently by different established methods. The true space group P3(1)21 was deduced, and a molecular replacement solution was obtained using the low resolution structure as template when searching in detwinned data.

Structural basis for activation of the thiamin diphosphate-dependent enzyme oxalyl-CoA decarboxylase by adenosine diphosphate.

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate-dependent enzyme that plays an important role in the catabolism of the highly toxic compound oxalate. We have determined the crystal structure of the enzyme from Oxalobacter formigenes from a hemihedrally twinned crystal to 1.73 A resolution and characterized the steady-state kinetic behavior of the decarboxylase. The monomer of the tetrameric enzyme consists of three alpha/beta-type domains, commonly seen in this class of enzymes, and the thiamin diphosphate-binding site is located at the expected subunit-subunit interface between two of the domains with the cofactor bound in the conserved V-conformation. Although oxalyl-CoA decarboxylase is structurally homologous to acetohydroxyacid synthase, a molecule of ADP is bound in a region that is cognate to the FAD-binding site observed in acetohydroxyacid synthase and presumably fulfils a similar role in stabilizing the protein structure. This difference between the two enzymes may have physiological importance since oxalyl-CoA decarboxylation is an essential step in ATP generation in O. formigenes, and the decarboxylase activity is stimulated by exogenous ADP. Despite the significant degree of structural conservation between the two homologous enzymes and the similarity in catalytic mechanism to other thiamin diphosphate-dependent enzymes, the active site residues of oxalyl-CoA decarboxylase are unique. A suggestion for the reaction mechanism of the enzyme is presented.

Oxalate-degrading Providencia rettgeri isolated from human stools.

BACKGROUND: Oxalate-degrading bacteria are thought to metabolize intestinal oxalate and thus decrease the urinary excretion of oxalate by reducing its intestinal absorption. METHODS: We have isolated several novel oxalate-degrading bacteria from human stools. Oxalate degrading bacteria were investigated to characterize their protein profiles with antibodies against oxalyl-coenzyme A decarboxylase (65 kDa) and formyl-coenzyme A transferase (48 kDa) purified from Oxalobacter formigenes. RESULTS: One of these isolates was identified as Providencia rettgeri, which showed two proteins (65 kDa and 48 kDa) on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) that were not found in non-oxalate-degrading P. rettgeri. Antibodies reacted with the 65 and 48 kDa proteins from the P. rettgeri strain on Western blotting. An Oxalobacter formigenes formyl-coenzyme A transferase gene probe reacted with chromosomal DNA from P. rettgeri on Southern blotting under high stringency conditions, while an Oxalobacter formigenes oxalyl-coenzyme A decarboxylase gene probe did not react under the same conditions. CONCLUSIONS: The mechamism of oxalate degradation by P. rettgeri appears to be similar to that of Oxalobacter formigenes. This is the first report of a facultative oxalate-degrading organism that is one of the Enterobacteriaceae.

Kinetic and mechanistic characterization of the formyl-CoA transferase from Oxalobacter formigenes.

Oxalobacter formigenes is an obligate anaerobe that colonizes the human gastrointestinal tract and employs oxalate breakdown to generate ATP in a novel process involving the interplay of two coupled enzymes and a membrane-bound oxalate:formate antiporter. Formyl-CoA transferase is a critical enzyme in oxalate-dependent ATP synthesis and is the first Class III CoA-transferase for which a high resolution, three-dimensional structure has been determined (Ricagno, S., Jonsson, S., Richards, N., and Lindqvist, Y. (2003) EMBO J. 22, 3210-3219). We now report the first detailed kinetic characterizations of recombinant, wild type formyl-CoA transferase and a number of site-specific mutants, which suggest that catalysis proceeds via a series of anhydride intermediates. Further evidence for this mechanistic proposal is provided by the x-ray crystallographic observation of an acylenzyme intermediate that is formed when formyl-CoA transferase is incubated with oxalyl-CoA. The catalytic mechanism of formyl-CoA transferase is therefore established and is almost certainly employed by all other members of the Class III CoA-transferase family.

Crystallization and preliminary crystallographic analysis of formyl-CoA tranferase from Oxalobacter formigenes.

Formyl-CoA transferase from Oxalobacter formigenes has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of formyl-CoA transferase were grown at 293 K using polyethylene glycol 4000 as a precipitant. The diffraction pattern of flash-frozen crystals at 100 K extends to 2.2 A resolution with synchrotron radiation (lambda = 0.933 nm). The crystals are tetragonal and belong to space group I4, with unit-cell parameters a = b = 151.44, c = 99.49 A. The asymmetric unit contains one dimer and the solvent content is 53%. Formyl-CoA transferase was crystallized both as the apoenzyme and as its complex with coenzyme A.

Oxalate-degrading enzymes from Oxalobacter formigenes: a novel device coating to reduce urinary tract biomaterial-related encrustation.

BACKGROUND AND PURPOSE: The long-term placement of biomaterials within the urinary tract is limited by the development of encrustation. In a noninfected urinary environment, encrustation often results from the deposition of calcium oxalate on the biomaterial surface. There is an association between the absence of Oxalobacter formigenes, a commensal colonic bacterium capable of degrading oxalate, and calcium oxalate stone formation. This pilot study was designed to evaluate several oxalate-degrading enzymes produced by O. formigenes as a potential biomaterial coating to reduce urinary tract encrustation. MATERIALS AND METHODS: Circular silicone disks of 6-mm diameter were incubated for 48 hours in oxalylcoenzyme A decarboxylase (OXC), formyl-coenzyme A transferase (FRC), and coenzyme A, while control disks were incubated in distilled water. The adsorption of OXC and FRC was assessed using enhanced chemiluminescence (ECL) and atomic force microscopy (AFM). Coated and uncoated disks (20 of each) were implanted in the bladders of 40 female New Zealand White rabbits. After 30 days, the disks were recovered, and the degree of encrustation on the polymer surface was evaluated utilizing dry weight measurement, calcium atomic absorption spectroscopy (AAS), and scanning electron microscopy/energy-dispersive X-ray analysis (SEM/EDX). RESULTS: Both ECL and AFM demonstrated coating of the silicone disks with OXC and FRC. The mean dry weights of the coated and control disks following explantation were 0.591 +/- 0.438 g and 0.747 +/- 0.428 g, respectively (P = 0.307). The mean weight of calcium on the coated and control disks, as determined by AAS, was 154.1 +/- 96.25 mg and 258 +/- 181.35 mg, respectively (P = 0.008). CONCLUSIONS: The use of oxalate-degrading enzymes from O. formigenes to coat urinary biomaterials represents a novel paradigm to reduce biomaterial-related encrustation. Coating of silicone with oxalate-degrading enzymes from O. formigenes results in a modest reduction in encrustation with no apparent toxicity. Further studies are warranted.